User:Andy Maloney/Notebook/Lab Notebook of Andy Maloney/2010/01/07/Surface passivation idea
I'm going to rehash the idea behind this project for my own edification. The whole idea behind understanding why people use casein as a surface passivator for gliding motility assays was to ultimately get rid of it. Casein is a globular protein that cannot be crystallized and thus we do not have an understanding of what it looks like besides its globular nature. Not to mention that casein comes from mammals and to use it, one has to purify it from mammal milk which comes with a whole slew of different issues. I should note that you can also make E. coli produce casein but again, that has other problems associated to it as well. Casein also was evolutionarily designed as a vehicle to transport calcium to growing mammals. Calcium is a known depolymerizer for microtubules so not using a big black box that may have calcium in it could be a good thing.
Initially I thought that casein was just a "blanket" of stuff that prevented the kinesin from coming in contact with glass. With this belief, I set out to try lipid films on glass in my gliding motility assays. After a large number of failed experiments that included: spin coated lipids, liposomes, and bilayers, I moved to using more complicated lipids. Again, this was all a failure. The one that made me give up completely was an 18:0 PE. I just couldn't get this to do anything. I did learn that when you are using lipids, you basically always want to use them in chloroform. Anything else and the possibility of failure for what you want to do increases. This is just my experience though.
After trying to work with lipids, I had to abandon this work because I had to start experiments using heavy water and gliding motility assays. These experiments of course worked spectacularly. I have plenty of data and am now awaiting tracking software to analyze it.
While I wait for the software, I've decided to return to investigating surface passivation for gliding motility assays. Only this time, I started to read more about kinesin and casein.
Verma et. al. showed that the different constituents of casein will support gliding motility assays. This on its own is a pretty awesome result but, I never thought about what it was that was the same between all the casein groups that allowed them to support motility.
I first looked into kappa-casein and what we know about its structure and the chemicals that make it up. It turns out that kappa-casein has carbohydrate tail groups that have a sugar group on them called a sialic acid. Sialic acid turns out to be a recognition site for things called lectins, which are basically adhesion molecules. After reading more, I rediscovered what kinectin is. Kinectin is a lectin that binds kinesin on the Golgi apparatus. This made me hope that casein somehow had similar binding properties as kinectin does to kinesin, and that kinesin was binding to the casein in the same way it does to kinectin. Unfortunately, kinectin binds the tail group of kinesin and I am using a truncated version of kinesin that does not have a tail group.
But, I didn't stop on the trail about sialic acid. I then continued to look into if sialic acid would recognize the stalk of kinesin somehow. From my brief examination of this idea, I believe that Collins et. al. describe that sialic acid does recognize and will "bind" to amino acids. Thus I looked at the stalk of kinesin and saw that the amino acids of the stalk, do have the possibility of binding to sialic acid.
At this point in time, I have only looked at kappa-casein and its sialic acid content. After some searching, I was able to find a paper that does show that alpha, beta, whole, and kappa casein all have sialic acid content, reference. This could mean that gliding motility assays work because of sialic acid (which is on the casein) binding to the kinesin stalk.
- Andy Maloney 18:40, 7 January 2010 (EST): Update - I did postulate that if it is the sialic acid stuff, then something like BSA should also work as a surface passivator. It turns out that Bohm uses BSA as the surface passivator. Hmmm, this idea is starting to look a bit more promising.
So, this is the new idea: determine if sialic acid is the way in which kinesin is prevented from coming in contact with glass. To do this, I've come up with a few ideas.
- Get some alpha-casein and beta-casein (we still have plenty of kappa and whole casein). Determine if all the caseins, alpha, beta, kappa, and whole will support gliding motility assays.
- If I can reproduce all the results of Verma, then I'd like to know the sialic acid content for each of the caseins. This is pricey, but there is a kit that will determine the sialic acid content. I think I'm definitely going to want this if I can get the okay for this experiment.
- If indeed all the casein do have sialic acid content, I will want to get some synthetic sialic acid and see if motility works with that. If not, then it may be because the sialic acid is not large enough to prevent the kinesin motor domains from getting close to the glass.
- If it does work, then I will need to show that it is indeed the sialic acid that is supporting motility. To do this, I will need to get rid of the sialic acid on all the caseins and try a motility assay. I can do this by cleaving the sialic acid with neuraminidase. This paper describes how they did it. I need to read it more closely.
- Andy Maloney 22:41, 7 January 2010 (EST): Update - I should note that the best case scenario would be if I could cap the sialic acid with a chemical I can wash away. That way I can definitively show that it is the sialic acid.
- If it turns out that synthetic sialic acid doesn't work, then I will want to try a glycolipid that is a lipid molecule that does have a sialic acid group on its head.